This project studies the structure and function of molecules in the Cys-loop receptor superfamily: the muscle nicotinic acetylcholine receptor (nAChR), the neuronal alpha4beta2 nAChR, and the serotonin 5-HT3 receptor. Hypothesis 1 states that three events occur in the following sequence at the agonist binding site, (a) The charged amine / ammonium group of the agonist is attracted to the site by a monopole-monopole interaction with fixed negative.charges on side chains, (b) For agonists with an amino group (not a quaternary ammonium group), this interaction is stabilized by an H-bond to the backbone carbonyl of the 149-150 peptide bond, (c) The earliest conformational change places the agonist in a cation-pi interaction at tryptophan alpha149. Hypothesis 2 states that ye M2-M3 linker undergoes a change in backbone conformation during gating. Hypothesis 3 states that during channel activation, the upper M2 helix of all five subunits re-orients with respect to neighboring helices. Hypothesis 4 states that the dynamic, history- dependent functional interaction between alpha4beta2 and P2X2 receptors occurs via the beta2-M3-M4 loop and the P2X2 C-terminal tail. Hypotheses 1 and 2 will be tested with macroscopic and single-channel electrophysiological assessments of receptors bearing unnatural amino-acid side chains and unnatural backbone linkages. Hypotheses 1, 3, and 4 will be tested in measurements based on direct fluorescence of tethered probes, fluorescence resonance energy transfer (FRET), and lanthanide-based resonance energy transfer (LRET). The resulting knowledge about acetylcholine receptors and 5-HT3 receptors may provide both pathophysiological insights and better drug therapies for health challenges including smoking cessation, Parkinson's disease, Alzheimer's disease, pain, Crohn's disease, sudden infant death syndrome, attention deficit disorder, autosomal dominant nocturnal frontal lobe epilepsy, and schizophrenia.